Project Summary/Abstract With annual mortality on dialysis of over 20%, kidney transplantation is the treatment of choice amongst those with end stage renal disease, however, while short term graft survival is improving, long term graft survival remains stagnant with antibody mediated rejection (ABMR) as its leading cause. Endothelial cells are the primary targets of injury in ABMR and the concept of including markers reflecting endothelial activation (endothelial- associated transcripts or ENDATs) is now included in its diagnostic criteria. Being that a late diagnosis often means suboptimal response to therapy, there is an urgent need to identify the mechanism by which endothelial injury occurs in ABMR to improve diagnostic and treatment strategies. Krppel-like factor (KLF4), a zinc-finger transcription factor exhibited the highest expression by microarray in kidney biopsies from patients with ABMR as compared to other types of graft dysfunction. In recent years, the critical role of KLF4 mediating anti- inflammatory gene expression in endothelial cells has been demonstrated in the cardiovascular system. In addition, KLF4 has been shown to inhibit cytokine induced signal transduction and activator of transcription (STAT3) signaling, a potential mechanism by which its anti-inflammatory effects may be mediated. However, the role of KLF4 in the renal endothelial response to injury is less well delineated. Based on these findings and preliminary data, our central hypothesis is that expression of KLF4 is required to prevent antibody-mediated endothelial injury by inhibition of STAT3 signaling and will be investigated by the following specific aims: 1) Test the hypothesis that endothelial-specific expression of KLF4 is required to prevent antibody mediated endothelial injury and 2) Determine the mechanism by which KLF4-STAT3 signaling is critical to preventing endothelial injury in ABMR. Under the first aim (subaim 1A), the protective role of KLF4 will be determined using a model of antibody induced injury in both stable knockdown (KLF4-shRNA) and overexpression (KLF4-ORF) human endothelial cell lines and appropriate controls. In subaim 1B, antibody mediated endothelial injury will be induced in mice with endothelial-specific Klf4 deletion and controls using the Cre-recombinase system to determine whether the loss of Klf4 accelerates endothelial damage. For aim 1, extent of endothelial injury will be determined by measuring the expression of ENDATs involved in adhesion and inflammation, as well as activation of STAT3 signaling by RT-PCR and western blot. Under the Aim 2 (subaim 2A), the mechanism by which KLF4 inhibits STAT3 signaling to prevent antibody-mediated endothelial injury will be elucidated by co-immunoprecipitation studies. In subaim 2B, flow cytometry will be used to isolate circulating endothelial cells from peripheral blood samples from patients with ABMR to evaluate whether the pattern of expression of KLF4 and STAT3 targets (IL- 6, ICAM-1) correlates with extent of endothelial injury on concurrent biopsy. This proposal will fill this current gap in the field by utilizing an integrative framework of cell culture and murine models to ascertain the mechanism by which regulation of KLF4-STAT3 signaling is critical to the prevention of endothelial injury in ABMR.